J. E. Seegmiller

Measurement of free nucleotides in cultured human lymphoid cells using high pressure liquid chromatography.

Abstract A method for the separation and quantitation of purine and pyrimidine nucleotides is described using high pressure liquid chromatography on a pellicular anion exchange resin. The method has been applied to extracts of cultured human lymphoid cell lines. An analysis can be carried out on 1−2 × 106 cells in 150 min. The soluble intracellular nucleotides appeared to increase rapidly in amount after subculture, but this was probably more related to increase in cell size than to increasing intracellular concentrations of nucleotides. No differences were found in soluble purine nucleotide levels between normal cells and cells deficient in hypoxanthine-guanine phosphoribosyltransferase. However, the enzyme-deficient cells had higher concentrations of soluble pyrimidine nucleotides, an unexpected finding. Growth of normal cells was inhibited by 5 × 10−5 or 10−4 m guanine or guanosine. This was associated with a marked expansion of the GTP pool with a marked decrease of the adenine and pyrimidine nucleotides. In HGPRT-deficient cells, guanine or guanosine produced no change in either growth or the intracellular concentrations of purine and pyrimidine nucleotides.

Increases in Purine Excretion and Rate of Synthesis by Drugs Inhibiting IMP Dehydrogenase or Adenylosuccinate Synthetase Activities

Hershfield reported an assay system devoid of hypoxanthine for evaluating the rate of purine synthesis de novo based on rates of [14C]-formate incorporation into intracellular purine components of lymphoblasts and into purines excreted into the culture medium. Using this system normal and in vitro selected, biochemically-characterized HPRT* deficient lines were found to have similar rates of total purine synthesis; however, these cell lines differed markedly in the excretion of newly-formed purine into the medium (1,2).

Purine biosynthesis in Chinese hamster cell mutants and human fibroblasts partially deficient in adenylosuccinate lyase.

Adenylosuccinate lyase (ASMP lyase, EC4.3.2.2) is the terminal enzyme j in de novo adenylate synthesis and as such acts to convert adenylosuccinate (AS) monophosphate to adenosine monophosphate. This enzyme also participates in the de novo synthesis of IMP converting 5′-phosphoribosyl-4-(N-succino carboxamide)-5-aminoimidazole (SAICAR) to 5′-phosphoribosyl-5-amino-4-imidazolecarboxamide (AICAR). Recently Jaeken, et al. described three patients with neurologic dysfunctions including infantile autism syndrome (1) who had elevated levels of AS and a compound tentatively identified as SAICA riboside in urine, blood, and cerebral spinal fluid. These patients have a markedly decreased but varying activity of ASMP lyase in various tissues studied. Deficiency of ASMP lyase has previously been described in mutagenized and nutrient-selected Chinese Hamster Ovary fibroblasts (CHO-Ade I cells) (2,3). These cells excrete excess ASMP and SAICAR into the culture medium. In the present study of purine nucleotide metabolism of CHO-Ade I cells and fibroblasts from a AS and SAICA riboside over-producing patient we examined the effect of ASMP lyase deficiency on branchpoint enzyme activities and on de novo synthesis by comparison to the control cell lines, wild type parent cells (CHO-K1) and fibroblasts from age-matched normal individuals, respectively.

Purine and Pyrimidine Nucleotide Concentrations in Cells with Decreased Hypoxanthine-Guanine-Phosphoribosyltransferase (HGPRT) Activity

1. In order to investigate the role of purine ribonucleotides in the regulation of de novo purine synthesis in living human cells deficient in HGPRT, intracellular ribonucleotide concentrations have been measured in HGPRT lymphoblasts, fibroblasts and erythrocytes and in appropriate HGPRT controls by high pressure liquid chromatography. 2. Purine Purine ribonucleotide concentrations were not reduced in HGPRT cells, supporting the hypothesis that accelerated purine biosynthesis de novo results from increased availability of PP-ribose-P and not from altered feedback regulation by purine ribonucleotides in HGPRT deficient cells. 3. Striking increases in intracellular concentrations of some pyrimidine nucleotides and nucleotide sugars were detected in HGPRT lymphoblasts and erythrocytes, but not in fibroblasts. 4. The possibiiity that this abnormality of pyrimidine metabolism might result from coordinate regulation of purine and pyrimidine synthesis de novo by PP-ribose-P was not substantiated by measurements of rates of pyrimidine synthesis and experimental elevation of intracellular concentrations of PP-ribose-P following incubation of cells with inorganic phosphate.

Experience with Detection of Heterozygous Carriers and Prenatal Diagnosis of Lesch-Nyhan Disease

For the purpose of genetic counselling, considerable efforts have been expended on the development of reliable methods necessary to identify the carriers of the Lesch-Nyhan syndrome, as well as to test in utero the sex of the fetus and its hypoxanthine phosphoribosyl transferase (HPRT, E.C. 2.4.2.8) activity.

Interactions between Energy Metabolism and Adenine Nucleotide Metabolism in Human Lymphoblasts

Adenine nucleotides play an important role in the transfer of chemical energy for metabolic processes. In addition, adenine nucleotide degradation can be a major source of purine bases formed during certain kinds of metabolic stress. These two properties of adenine nucleotides may be related. Fructose and 2-deoxyglucose can produce elevated levels of purines in vivo (1), in perfused organs (2), and in cultured cells (3,4). The mechanism of nucleotide degradation caused by fructose or 2-deoxyglucose involves the utilization of ATP to form a slowly metabolized hexose phosphate which accumulates and decreases the intracellular concentration of inorganic phosphate (1–4). AMP deaminase, which is normally inhibited by phosphate, becomes more active when the phosphate concentration decreases and adenine nucleotides are broken down by the reactions: AMP → IMP + NH3; IMP → inosine + PO 4 2- . There also exists another pathway for adenine nucleotide degradation catalyzed by the enzymes purine 5′-nucleotidase and adenosine deaminase: AMP → adenosine + PO 4 2- ; adenosine → inosine + NH3.

Adenosine and guanosine metabolism during phytohemagglutinin induced transformation of human lymphocytes.

Reports of the human genetic deficiency of adenosine deaminase activity in association with severe combined immunodeficiency disease (1,2) suggested adenosine deaminase activity may be necessary for lymphocyte response. We have examined the effect of adenosine and inhibitors of adenosine deaminase activity on lymphocyte transformation. Work in this laboratory has shown the inhibition of lymphocyte transformation by adenosine to be potentiated by inhibitors of adenosine deaminase activity in concanavalin A (3) or phytohemagglutinin (PHA) (4) stimulated human lymphocytes. The report of purine nucleoside phosphosphorylase deficiency in association with defective T cell immunity (5) adds further impetus to the investigation of purine nucleoside metabolism in lymphocytes. The present report describes changes in adenosine and guanosine metabolism in both lysates and intact cells during PHA-induced transformation of human lymphocytes.

Clinical and Biochemical Correlates of A New HPRT Mutation

In the biochemical analysis of patients with Lesch-Nyhan disease the activity of HPRT found in cell lysates does not always correlate with HPRT activity in the intact cell (1–3). Neither analysis consistently predicts the degree of neurologic impairment in the patient (4,5).

Regulation of Human Lymphoblast Ecto-5′-Nucleotidase by Zinc

Ecto-5′-nucleotidase (5′NT) catalyzes the degradation of purine nucleotide-5′-monophosphates to nucleosides and inorganic phosphate; it is an integral plasma membrane enzyme of most mammalian cells and widely used as a membrane marker. Although the physiological role of 5′NT is still mostly obscure, decreased 5′NT activity was found to be associated with a variety of immunological disorders and lymphoproliferative diseases (1–6). 5′NT was suggested to provide a marker for B- and T-lymphocyte maturation (5,7).